Apoptosis is the regulated death and removal of cells from the body. Defects in apoptosis are associated with the pathogenesis of multiple diseases, including neurodegenerative disorders. Thus, elucidating the mechanisms which regulate this type of cell death at the molecular level is of paramount importance to understanding disease progression. Apoptosis is regulated by specific signaling pathways which ensure that inappropriate cell death does not occur. Modulation of intracellular calcium concentration via channels, pumps, and transporters is crucial to the execution of the apoptotic program. The inositol 1,4,5- trisphosphate receptor (IP3R) calcium channel contributes to calcium release during apoptosis. There have been several mechanisms suggested for the activation of this channel during apoptosis, but an integrated model which describes IP3R activation both early and late in apoptotic signaling is lacking. In this proposal we will examine the molecular mechanisms by which this channel is activated in response to cytotoxic stress and death-receptor stimuli. In Aim 1 we will determine the requirement of phospholipase C activation and subsequent IP3 generation for IP3R-dependent apoptosis. We will use genetic knockout and RNAi to elucidate the molecular requirement of IP3 generation for calcium release and cell death. In Aim 2 we will determine the requirement of cytochrome c binding to the channel and subsequent augmentation of calcium release, and the temporal relationship of this event to IP3 binding to the channel. We will utilize several novel systems including Drosophila and cytochrome c knockout cells and dominant negative peptides. In Aim 3 we will determine the requirement of caspase cleavage of the channel and the relationship of this event to IP3 generation and cytochrome c binding to the channel. We will use genetic knockouts of caspase-3/9 and IP3R and rescue studies to examine the relative contribution of caspase-3 cleavage for apoptotic calcium release and cell death. The aim of these studies is to understand the precise signaling mechanisms which regulate calcium release from IP3R channels during apoptosis, with the ultimate goal of identifying novel targets for therapeutic intervention.